GB 1886.174-2016 English PDF

GB 1886.174-2016: National food safety standard - Food additives - Enzyme preparation for food industry
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GB 1886.174-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Food Additives - Enzyme preparation for food industry
ISSUED ON: AUGUST 31, 2016
IMPLEMENTED ON: JANUARY 01, 2017
Issued by: National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions ... 4 
3 Product classification ... 4 
4 Technical requirements ... 4 
Appendix A Method for determining enzyme activity ... 7 
Appendix B Table for comparison of absorbance and α-amylase concentration
... 44 
National Food Safety Standard -
Food Additives - Enzyme preparation for food industry
1 Scope
This Standard applies to the enzyme preparation for food industry allowed by
GB 2760.
2 Terms and definitions
2.1 Enzyme preparation for food industry
Biological products with special catalytic functions for food processing, which
are obtained by directly extracting the edible or non-edible parts of animals or
plants, or by fermentation and extraction from traditional or genetically-modified
microorganisms (including but not limited to bacteria, actinomycetes, fungal
strains).
Note: Commercial enzyme preparation products allow the incorporation of ingredients
which are suitable for the product storage and use.
2.2 Enzyme activity
The ability of an enzyme to catalyze a particular reaction under certain
conditions, i.e., enzyme activity. It is a characteristic exclusive index for the
expression of enzyme preparation products.
2.3 Antibacterial activity
The ability to inhibit or kill microorganisms.
3 Product classification
According to the product form, it is classified into two types: solid dosage form
and liquid dosage form.
4 Technical requirements
4.1 Raw material requirements
Appendix A
Method for determining enzyme activity
A.1 General
The reagents and water used in this Standard, when no other requirements are
specified, refer to the analytical reagents and the Grade 3 water specified in
GB/T 6682. Standard solutions, impurity standard solutions, preparations, and
products used in the test, when no other requirements are specified, shall be
prepared in accordance with the provisions of GB/T 601, GB/T 602, and GB/T
603. The solution used in the test, when no solvent is specified, refers to the
aqueous solution.
A.2 Determination of α-amylase activity
A.2.1 α-amylase
An enzyme capable of hydrolyzing the α-1,4 glycosidic bond in the molecular
chain of starch, and cutting the starch chain into a short-chain dextrin and a
small amount of maltose and glucose to rapidly decrease the viscosity of the
starch.
A.2.2 α-amylase activity
A.2.2.1 Medium-temperature α-amylase activity unit
1 g of solid enzyme powder (or 1 mL of liquid enzyme), at 60 °C, pH 6.0,
liquefies 1 g of soluble starch in 1 h, i.e. 1 enzyme activity unit, expressed in
U/g (U/mL).
A.2.2.2 High-temperature resistant α-amylase activity unit
1 g of solid enzyme powder (or 1 mL of liquid enzyme), at 70 °C, pH 6.0,
liquefies 1 mg of soluble starch in 1 min, i.e. 1 enzyme activity unit, expressed
in U/g (U/mL).
A.2.3 Principle
The α-amylase preparation can randomly cut the α-1,4 glycosidic bond in the
starch molecular chain to short-chain dextrin of various lengths, a small amount
of maltose and glucose, so that the characteristic reaction of starch showing
bluish violet to iodine gradually disappears, showing a brownish red. The rate
at which the color disappears is related to the enzyme activity, whereby the
mL; USE a small amount of phosphate buffer to dissolve it thoroughly; carefully
POUR the supernatant into the volumetric flask. If there is residual residue,
ADD a small amount of phosphate buffer to fully grind; TRANSFER all the final
sample into the volumetric flask; USE phosphate buffer to dilute to the mark;
SHAKE well. USE four layers of gauze to filter and leave the filtrate for use.
Note: For the enzyme activity of the medium-temperature α-amylase enzyme solution to
be tested, the enzyme concentration is controlled in the range of 3.4 U/mL~4.5 U/mL.
For the high-temperature resistant α-amylase activity to be tested, the enzyme
concentration is controlled in the range of 60 U/mL~65 U/mL.
A.2.6.2 Determination
a) PIPETTE 20.0 mL of soluble starch solution into the test tube; ADD 5.00
mL of phosphate buffer; after shaking well, PLACE in a constant-
temperature water bath at 60 °C±0.2 °C (The high-temperature resistant
α-amylase preparation is placed at 70 °C±0.2 °C) to preheat for 8 min;
b) ADD 1.00 mL of diluted enzyme solution to be tested; TIME immediately;
SHAKE well; accurately react for 5 min;
c) Immediately USE an automatic pipette to pipette 1.00 mL of reaction
solution; ADD it to a test tube preloaded with 0.5 mL of hydrochloric acid
solution and 5.00 mL of dilute iodine solution; SHAKE well. USE 0.5 mL
of hydrochloric acid solution and 5.00 mL of dilute iodine solution as blanks;
at a wavelength of 660 nm, USE a 10 mm cuvette to rapidly determine the
absorbance (A). According to the absorbance, refer to the table of
Appendix B, to obtain the concentration of the test enzyme solution.
A.2.6.3 Result calculation
A.2.6.3.1 Enzyme activity of medium-temperature α-amylase preparation
The enzyme activity X1 of medium-temperature α-amylase preparation, in U/mL
or U/g, is calculated according to formula (A.1):
Where:
c - The concentration of the test enzyme sample, in U/mL or U/g;
n - The dilution factor of the sample.
The result obtained is expressed to integer.
The test result is based on the arithmetic mean of parallel determination results.
A.3.3.2 0.05 mol/L sodium thiosulfate standard titration solution.
A.3.3.3 0.1 mol/L iodine standard solution.
A.3.3.4 0.1 mol/L sodium hydroxide solution.
A.3.3.5 2 mol/L sulfuric acid solution: PIPETTE 5.6 mL of analytical
concentrated sulfuric acid (Relative density is 1.84); slowly ADD it to the
appropriate amount of water; after cooling, USE water to dilute to 100 mL;
SHAKE well.
A.3.3.6 200 g/L sodium hydroxide solution: WEIGH 20 g of sodium hydroxide;
USE water to dissolve and dilute to 100 mL.
A.3.3.7 20 g/L soluble starch solution: WEIGH 2 g±0.001 g of soluble starch;
then USE a small amount of water to mix thoroughly; slowly POUR into boiling
water; BOIL, stir until it is transparent; COOL; USE water to dilute to 100 mL.
This solution needs to be prepared on the same day.
Note: Soluble starch shall be special starch for analysis of enzyme preparation.
A.3.4 Instruments and equipment
A.3.4.1 Analytical balance: Accuracy is 0.2 mg.
A.3.4.2 Acidimeter: Accuracy is 0.01 pH.
A.3.4.3 Analytical balance: Accuracy is 0.2 mg.
A.3.4.4 Constant-temperature water bath: 40 °C±0.1 °C.
A.3.4.5 Continuous multi-gear distributor (pipette).
A.3.4.6 Magnetic stirrer.
A.3.5 Analytical procedures
A.3.5.1 Preparation of enzyme solution to be tested
A.3.5.1.1 Liquid enzyme: USE a continuous multi-gear distributor to accurately
pipette the appropriate amount of enzyme sample; TRANSFER it into a
volumetric flask; USE a buffer solution to dilute to the mark; SHAKE well for
determination.
A.3.5.1.2 Solid enzyme: USE a 50 mL small beaker to accurately weigh the
appropriate amount of enzyme sample, accurate to 1 mg. USE a small amount
of acetic acid-sodium acetate buffer solution to dissolve; USE a glass rod to
carefully grind. Carefully POUR the supernatant into a suitable volumetric flask;
dilute to 1000 mL.
A.4.4.8.2 Sodium lactate buffer (pH=3.0, suitable for acidic protease
preparations).
TAKE 4.71 g of lactic acid (80%~90%) and 0.89 g of sodium lactate (70%); ADD
water to 900 mL; STIR well. USE lactic acid or sodium lactate to adjust the pH
to 3.0±0.05; DILUTE to 1000 mL.
A.4.4.8.3 Boric acid buffer solution (pH=10.5, suitable for alkaline protease
preparations).
WEIGH 9.54 g of sodium borate, 1.60 g of sodium hydroxide; ADD 900 mL of
water; STIR well. USE 1 mol/L hydrochloric acid solution or 0.5 mol/L sodium
hydroxide solution to adjust the pH=10.5±0.05; DILUTE to 1000 mL.
A.4.4.9 Casein solution (10.0 g/L): WEIGH 1.000 g of standard casein (NICPBP
National Drug Reference Material), accurate to 0.001 g. After using a small
amount of sodium hydroxide solution (for the acidic protease preparation, USE
2 drops~3 drops of concentrated lactic acid) to moisten, ADD about 80 mL of
the corresponding buffer solution; HEAT and boil for 30 min in a boiling water
bath; STIR occasionally until the casein is completely dissolved. After cooling
to room temperature, TRANSFER to a 100 mL volumetric flask; USE a suitable
pH buffer solution to dilute to the mark. Before diluting to the mark, CHECK and
adjust the pH to the specified value of the corresponding buffer. This solution is
stored in the refrigerator and is valid for 3 days. Before use, reconfirm and
adjust the pH to the specified value.
Note: Casein from different sources or batch numbers has an effect on the test results. If
different casein is used as a substrate, before use, the results shall be compared
with the above standard casein.
A.4.4.9.1 L-tyrosine standard stock solution (100 μg/mL): Accurately WEIGH
0.1000 g±0.0002 g of L-tyrosine previously dried at 105 °C to a constant mass;
USE 60 mL of 1 mol/L hydrochloric acid solution to dissolve and dilute to 100
mL, i.e. 1 mg/mL tyrosine solution.
PIPETTE 10.00 mL of 1 mg/mL tyrosine solution; USE 0.1 mol/L hydrochloric
acid solution to dilute to 100 mL, to obtain 100 μg/mL L-tyrosine standard stock
solution.
Note: In addition to the above protease dissolution/dilution buffer systems, producers and
users can also explore the use of other suitable buffer systems.
A.4.5 Instruments and equipment
P9135 or equivalent, accurate to 0.1 mg); ADD water to dissolve; BOIL, cool,
and filter. Adjust the pH to 3.5; USE water to dilute to 100 mL; STORE in a
refrigerator for use. The use time is no more than 3 days.
Note: The pectin substrate has a great influence on the test. If pectin powders from
different sources or batch numbers are used, it shall perform a controlled test against
the former batch number.
A.5.4.2 Sodium thiosulfate standard solution: c (Na2S2O3)=0.05 mol/L.
A.5.4.3 Sodium carbonate standard solution: c (12 Na2CO3)=2 mol/L.
A.5.4.4 Iodine standard solution: c (12 I2)=0.1 mol/L.
A.5.4.5 2 mol/L sulfuric acid solution: TAKE 5.6 mL of concentrated sulfuric acid;
slowly ADD it to the appropriate amount of water; after cooling, USE water to
dilute to 100 mL; SHAKE well and set aside.
A.5.4.6 Soluble starch indicator solution (10 g/L).
A.5.4.7 0.1 mol/L citric acid-sodium citrate buffer (pH 3.5): WEIGH 14.71 g of
citric acid (C6H8O7 • H2O), 8.82 g of trisodium citrate (C6H5Na3O7 • 2H2O); ADD
950 mL of water to dissolve; adjust the pH to 3.5; USE water to dilute to 1000
mL.
A.5.5 Instruments and equipment
A.5.5.1 Colorimetric tube: 25 mL.
A.5.5.2 Constant-temperature water bath with heating device: Temperature
control accuracy is ±0.1 °C.
A.5.5.3 Iodine flask: 250 mL.
A.5.5.4 Burette: 25 mL.
A.5.6 Analytical procedures
A.5.6.1 Preparation of sample solution
USE a 50 mL beaker of known mass to weigh 1 g~2 g of enzyme powder
(accurate to 0.0001 g) or accurately pipette 1.00 mL. USE a small amount of
citric acid-sodium citrate buffer to thoroughly dissolve; USE a glass rod to grind;
carefully POUR the supernatant into a volumetric flask. If there is residual
residue, ADD a small amount of the above buffer to fully grind; TRANSFER all
The presence of 3-hydroxy-2-butanone (acetoin) and/or diacetyl in the sample
may result in a large test result.
The acetoin, during storage, is easy to form a dimer, thus affecting the test
results. However, if the measures described in the preparation steps are
followed to prevent, this method may still be used.
A.6.3.2 Principle
The α-acetolactate decarboxylase reacts with the substrate α-acetolactate to
decarboxylate to form acetoin. The acetoin, under alkaline conditions, reacts
with a mixture of naphthol and creatine to form a red product. By determining
the absorbance of the solution at 522 nm, the amount of acetoin produced by
the reaction can be obtained from the standard curve of acetoin; so that the
enzyme activity of α-acetolactate decarboxylase can be calculated.
A.6.3.3 Reagents and materials
A.6.3.3.1 MES (9.76 g/L)-sodium chloride (35.064 g/L)-polyoxyethylene lauryl
ether1) (1.52 mL/L) buffer: WEIGH 48.80 g of 2-[N-morpholino]ethanesulfonic
acid (MES) and 175.32 g of sodium chloride in a beaker; USE about 4.5 L of
water to dissolve. Then ADD 7.60 mL of 15% polyoxyethylene lauryl ether
solution; STIR well. USE about 1 mol/L sodium hydroxide solution to adjust the
pH to 6.00 ± 0.05. Then TRANSFER to a 5000 mL volumetric flask; USE water
to dilute to the mark; STIR well. This solution, at room temperature
(15 °C~20 °C), has a storage life of one week.
A.6.3.3.2 α-acetolactate substrate (2.00 mL/L): PIPETTE 100 μL of ethyl-2-
acetoxy-2- methyl-acetoacetic acid into a 50 mL volumetric flask; ADD 6.0 mL
of about 0.50 mol/L sodium hydroxide solution. After stirring for 20 min, ADD
buffer to about 40.0 mL; USE about 1 mol/L hydrochloric acid to adjust the pH
of the solution to 6.00±0.05. Then, USE buffer to dilute to the mark. This solution
is prepared before use.
A.6.3.3.3 Naphthol (10.0 g/L)/creatine (1.0 g/L) color developer: WEIGH 5.0 g
of 1-naphthol and 0.5 g of creatine into 500 mL volumetric flask; USE about 1
mol/L sodium hydroxide solution to dissolve and dilute to the mark. This solution
is prepared before use. During the preparation, it shall be protected from light
and be in ice bath.
Warning - 1-naphthol is flammable and toxic. It is irritating to the eyes and
mucous membranes. If swallowed or absorbed through the skin,
1 ) Brij®35 is a suitable commercially-available product. Products with the equivalent
analytical effect may also be used.
A.6.3.5.1 Preparation ...